Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Kinesins control male germ unit assembly for sperm delivery in Arabidopsis

Nature Plants volume 11, pages 1798–1809 (2025) Cite this article

Subjects

Abstract

Unlike flagellated sperm in animals, which use molecular motors for motility, immotile sperm cells of angiosperms rely on cytoplasmic transport within pollen tubes to reach female gametes for fertilization. However, the mechanism underlying sperm cell transport in angiosperms remains unknown. Since the 1970s it has been observed that the two sperm cells, or their progenitor generative cell, are transported together with the pollen vegetative nucleus as part of an aggregated structure called the male germ unit, which forms within the pollen cytoplasm. Here, using super-resolution and live-cell imaging, we show that two kinesins, HUG1 and HUG2, form a kinesin cage encasing a microtubule cage around the generative cell or sperm cells and vegetative nucleus, tethering them into a single unit during Arabidopsis pollen development. Loss of HUG proteins disrupts male germ unit organization, leading to failed sperm delivery and complete plant sterility. These findings uncover the genetic and cellular basis of male germ unit organization and highlight its essential role in sperm transport for plant fertilization.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Sperm cells and the vegetative nucleus are separated in hug1 hug2 double mutant pollen.
The alternative text for this image may have been generated using AI.
Fig. 2: HUG1 and HUG2 localize around the MGU.
The alternative text for this image may have been generated using AI.
Fig. 3: Assembly of the HUG cage depends on the microtubule cage.
The alternative text for this image may have been generated using AI.
Fig. 4: The CC2_Tail domain is required for VC–GC tethering.
The alternative text for this image may have been generated using AI.
Fig. 5: Working model of HUGs in MGU organization.
The alternative text for this image may have been generated using AI.

Similar content being viewed by others

Data availability

The data for the current study are available within the paper and Supplementary Information or from the corresponding authors upon request. Source data are provided with this paper.

References

  1. Lin, J. & Nicastro, D. Asymmetric distribution and spatial switching of dynein activity generates ciliary motility. Science 360, eaar1968 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Ortiz-Ramirez, C. et al. GLUTAMATE RECEPTOR-LIKE channels are essential for chemotaxis and reproduction in mosses. Nature 549, 91–95 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Rudall, P. J. & Bateman, R. M. Developmental bases for key innovations in the seed-plant microgametophyte. Trends Plant Sci. 12, 317–326 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Hackenberg, D. & Twell, D. The evolution and patterning of male gametophyte development. Curr. Top. Dev. Biol. 131, 257–298 (2019).

    Article  PubMed  Google Scholar 

  5. Dresselhaus, T., Sprunck, S. & Wessel, G. M. Fertilization mechanisms in flowering plants. Curr. Biol. 26, R125–R139 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hafidh, S. & Honys, D. Reproduction multitasking: the male gametophyte. Annu. Rev. Plant Biol. 72, 581–614 (2021).

    Article  CAS  PubMed  Google Scholar 

  7. Cheung, A. Y., Duan, Q., Li, C., James Liu, M. C. & Wu, H. M. Pollen–pistil interactions: it takes two to tangle but a molecular cast of many to deliver. Curr. Opin. Plant Biol. 69, 102279 (2022).

    Article  CAS  PubMed  Google Scholar 

  8. Sugi, N. et al. The peri-germ cell membrane: poorly characterized but key interface for plant reproduction. Nat. Plants 10, 1607–1609 (2024).

    Article  PubMed  Google Scholar 

  9. Brewbaker, J. L. The distribution and phylogenetic significance of binucleate and trinucleate pollen grains in the angiosperms. Am. J. Bot. 54, 1069–1083 (1967).

    Article  Google Scholar 

  10. Russell, S. D. & Cass, D. D. Ultrastructure of the sperms of Plumbago zeylanica. Protoplasma 107, 85–107 (1981).

    Article  Google Scholar 

  11. McCue, A. D., Cresti, M., Feijó, J. A. & Slotkin, R. K. Cytoplasmic connection of sperm cells to the pollen vegetative cell nucleus: potential roles of the male germ unit revisited. J. Exp. Bot. 62, 1621–1631 (2011).

    Article  CAS  PubMed  Google Scholar 

  12. Mogensen, H. L. The male germ unit: concept, composition, and significance. Int. Rev. Cytol. 140, 129–147 (1992).

    Article  Google Scholar 

  13. McConchie, C. A., Jobson, S. & Knox, R. B. Computer-assisted reconstruction of the male germ unit in pollen of Brassica campestris. Protoplasma 127, 57–63 (1985).

    Article  Google Scholar 

  14. Dumas, C., Knox, R. B. & Gaude, T. The spatial association of the sperm cells and vegetative nucleus in the pollen grain of Brassica. Protoplasma 124, 168–174 (1985).

    Article  Google Scholar 

  15. Palevitz, B. A. Relationship between the generative cell and vegetative nucleus in pollen tubes of Nicotiana tabacum. Sex. Plant Reprod. 6, 1–10 (1993).

    Article  Google Scholar 

  16. Dumas, C., Knox, R. B., McConchie, C. A. & Russell, S. D. Emerging physiological concepts in fertilisation. What’s New in Plant Physiol. 15, 17–20 (1984).

    Google Scholar 

  17. Zhou, X. & Meier, I. Efficient plant male fertility depends on vegetative nuclear movement mediated by two families of plant outer nuclear membrane proteins. Proc. Natl Acad. Sci. USA 111, 11900–11905 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Motomura, K. et al. Persistent directional growth capability in Arabidopsis thaliana pollen tubes after nuclear elimination from the apex. Nat. Commun. 12, 2331 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Schattner, S., Schattner, J., Munder, F., Höppe, E. & Walter, W. J. A tug-of-war model explains the saltatory sperm cell movement in Arabidopsis thaliana pollen tubes by kinesins with calponin homology domain. Front. Plant Sci. 11, 601282 (2020).

    Article  PubMed  Google Scholar 

  20. Heslop-Harrison, J. & Heslop-Harrison, Y. Conformation and movement of the vegetative nucleus of the angiosperm pollen tube: association with the actin in cytoskeleton. J. Cell Sci. 93, 299–308 (1989).

    Article  Google Scholar 

  21. Åström, H., Sorri, O. & Raudaskoski, M. Role of microtubules in the movement of the vegetative nucleus. Sex. Plant Reprod. 8, 61–69 (1995).

    Article  Google Scholar 

  22. Heslop-Harrison, J. & Heslop-Harrison, Y. Cytoskeletal elements, cell shaping and movement in the angiosperm pollen tube. J. Cell Sci. 91, 49–60 (1988).

    Article  Google Scholar 

  23. Wang, X. et al. Distinct functions of microtubules and actin filaments in the transportation of the male germ unit in pollen. Nat. Commun. 15, 5448 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Borges, F. et al. FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei. Plant Methods 8, 44 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Abe, A. et al. Genome sequencing reveals agronomically important loci in rice using MutMap. Nat. Biotechnol. 30, 174–178 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Nebenführ, A. & Dixit, R. Kinesins and myosins: molecular motors that coordinate cellular functions in plants. Annu. Rev. Plant Biol. 69, 329–361 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Ali, I. & Yang, W. C. Why are ATP-driven microtubule minus-end directed motors critical to plants? An overview of plant multifunctional kinesins. Funct. Plant Biol. 47, 524–536 (2020).

    Article  CAS  PubMed  Google Scholar 

  28. Gicking, A. M., Swentowsky, K. W., Dawe, R. K. & Qiu, W. Functional diversification of the kinesin-14 family in land plants. FEBS Lett. 592, 1918–1928 (2018).

    Article  CAS  PubMed  Google Scholar 

  29. Miki, H., Okada, Y. & Hirokawa, N. Analysis of the kinesin superfamily: insights into structure and function. Trends Cell Biol. 15, 467–476 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Honys, D. & Twell, D. Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol. 5, R85 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Palanivelu, R. & Preuss, D. Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro. BMC Plant Biol. 6, 7 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Chebli, Y., Kroeger, J. & Geitmann, A. Transport logistics in pollen tubes. Mol. Plant 6, 1037–1052 (2013).

    Article  CAS  PubMed  Google Scholar 

  33. Cai, G. & Cresti, M. Organelle motility in the pollen tube: a tale of 20 years. J. Exp. Bot. 60, 495–508 (2009).

    Article  CAS  PubMed  Google Scholar 

  34. Kawashima, T. et al. Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana. eLife 3, e04501 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Chen, S. Y. et al. Osmoregulation determines sperm cell geometry and integrity for double fertilization in flowering plants. Mol. Plant 15, 1488–1496 (2022).

    Article  CAS  PubMed  Google Scholar 

  36. Boavida, L. C., Qin, P., Broz, M., Becker, J. D. & McCormick, S. Arabidopsis tetraspanins are confined to discrete expression domains and cell types in reproductive tissues and form homo- and heterodimers when expressed in yeast. Plant Physiol. 163, 696–712 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gilles, L. M. et al. Lipid anchoring and electrostatic interactions target NOT-LIKE-DAD to pollen endo-plasma membrane. J. Cell Biol. 220, e202010077 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhu, C. & Dixit, R. Functions of the Arabidopsis kinesin superfamily of microtubule-based motor proteins. Protoplasma 249, 887–899 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Wang, Q. & Huang, S. Visualization of microtubule organization and dynamics in living Arabidopsis embryonic cells. Mol. Plant 7, 1397–1401 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Tamura, K. et al. Myosin XI-i links the nuclear membrane to the cytoskeleton to control nuclear movement and shape in Arabidopsis. Curr. Biol. 23, 1776–1781 (2013).

    Article  CAS  PubMed  Google Scholar 

  41. Aström, H. Acetylated α-tubulin in the pollen tube microtubules. Cell Biol. Int. Rep. 16, 871–881 (1992).

    Article  PubMed  Google Scholar 

  42. Verhey, K. J. & Ohi, R. Causes, costs and consequences of kinesin motors communicating through the microtubule lattice. J. Cell Sci. 136, jcs260735 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Li, S., Zhou, L. Z., Feng, Q. N., McCormick, S. & Zhang, Y. The C-terminal hypervariable domain targets Arabidopsis ROP9 to the invaginated pollen tube plasma membrane. Mol. Plant 6, 1362–1364 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Shi, L., Mogensen, H. L. & Zhu, T. Dynamics of nuclear pore density and distribution patterns within developing pollen: implications for a functional relationship between the vegetative nucleus and the generative cell. J. Cell Sci. 99, 115–120 (1991).

    Article  Google Scholar 

  45. Wang, Z.-P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  PubMed  Google Scholar 

  47. Wang, W. et al. A non-defensin peptide NPA1 attracts pollen tube in Arabidopsis. Seed Biol. 3, e003 (2024).

    Article  Google Scholar 

  48. Long, Y. et al. In vivo FRET–FLIM reveals cell-type-specific protein interactions in Arabidopsis roots. Nature 548, 97–102 (2017).

    Article  CAS  PubMed  Google Scholar 

  49. Wu, Y. et al. Arabidopsis FIMBRIN5, an actin bundling factor, is required for pollen germination and pollen tube growth. Plant Cell 22, 3745–3763 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Waadt, R. et al. Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J. 56, 505–516 (2008).

    Article  CAS  PubMed  Google Scholar 

  51. Yoo, S.-D., Cho, Y.-H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31991203 and 32130032 to W.-C.Y.; 32425009 and 32170343 to H.-J.L.), the National Key Research and Development Program of China (2022YFF1003500 to H.-J.L.), the Strategic Priority Research Program of the Chinese Academy of Science (XDB1090000 to H.-J.L.) and CAS Project for Young Scientists in Basic Research (YSBR-078 to H.-J.L.). We thank S. Huang (Tsinghua University) for GFP–MBD seeds, J. Becker (Instituto Gulbenkian de Ciência) for MGH3pro:MGH3–GFP ACT11pro:H2B–RFP plants and the Bio-Imaging Facility (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for confocal microscopy.

Author information

Author notes

  1. These authors contributed equally: Shu Chang, Iftikhar Ali, Peng-Min Zhou.

Authors and Affiliations

  1. State Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Shu Chang & Hong-Ju Li

  2. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Iftikhar Ali, Peng-Min Zhou, Hua-Mao Wu, Dong-Qiao Shi & Wei-Cai Yang

  3. School of Life Sciences, Tsinghua University, Beijing, China

    Haisu Cheng & Xin Liang

  4. Yazhouwan National Laboratory, Sanya, China

    Wei-Cai Yang

Authors
  1. Shu Chang
    View author publications

    Search author on:PubMed Google Scholar

  2. Iftikhar Ali
    View author publications

    Search author on:PubMed Google Scholar

  3. Peng-Min Zhou
    View author publications

    Search author on:PubMed Google Scholar

  4. Haisu Cheng
    View author publications

    Search author on:PubMed Google Scholar

  5. Xin Liang
    View author publications

    Search author on:PubMed Google Scholar

  6. Hua-Mao Wu
    View author publications

    Search author on:PubMed Google Scholar

  7. Dong-Qiao Shi
    View author publications

    Search author on:PubMed Google Scholar

  8. Hong-Ju Li
    View author publications

    Search author on:PubMed Google Scholar

  9. Wei-Cai Yang
    View author publications

    Search author on:PubMed Google Scholar

Contributions

W.-C.Y. and H.-J.L. conceived of and directed the study. S.C., I.A. and P.-M.Z. performed the experiments and analysed the data. D.-Q.S. and H.-M.W. helped with phenotypic observation. H.C. and X.L. helped with protein expression and purification. H.-J.L. and W.-C.Y. analysed the data and wrote the paper. All authors read and agreed to submission of the final version of the paper.

Corresponding authors

Correspondence to Hong-Ju Li or Wei-Cai Yang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Plants thanks Giampiero Cai, Iris Meier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Expression pattern and phylogenetic analysis of HUG proteins.

(a) The distance between vegetative nucleus and sperm cells are longer in the site-mutated hug1 mutant than in WT. Scale bar, 10 μm. (b) Expression level of HUG1 and HUG2 in select cell types from transcriptome data published in30. UNM, microspores; BCP, bicellular pollen; TCP, tricellular pollen; MPG, mature pollen; COT, cotyledon; LEF, leaf; PET, petiole; STM, stem; ROT, root; RHZ, root-hair zone; SUS, suspension cell. (c) Phylogenetic tree of the closest HUG homologues in select terrestrial plant species. Sequences were retrieved from Phytozome or NCBI and were aligned by CLUSTAL W. The phylogenetic tree was constructed with MEGA7 using the neighbor-joining method with 1,000 bootstraps.

Extended Data Fig. 2 Characterization of Arabidopsis hug1 and hug2 mutants.

(a) Genomic structure (top) and summary of the hug1 mutations generated by, EMS mutagenesis or CRISPR–Cas9 (bottom). (b) Genomic structure (top) and summary of the hug2 mutations generated by CRISPR–Cas9 (bottom) or T-DNA insertions. (c-d) Seed set in siliques of WT and hug1 and hug2 single and double mutants. Scale bar, 1 mm. (e-f) Quantification of seed set shown as in (c–d). In (e), n = 3,282, 3,701, 3,479, 3,493, 4,081 and 3,537 ovules for genotypes from left to right. Data are the mean ± S.D. In (f), n = 1,523, 1,546, 1,621, 1,554, 1,601 and 1,479 ovules. Data are the mean ± S.D. Two-sided Students’ t-test, ***P = 2.31 × 10−5 (hug1-2 hug2-2) and ***P = 6.88 × 10−6 (♀ WT × ♂ hug1-2 hug2-2) compared with WT. (g) Pollen-germination rate for WT and hug double mutants. Data are the mean ± S.D. Two-sided Students’ t-test, n.s. non-significant. P = 0.9268 n = 300 grains for each sample. (h–i) Successful pollen tube entry into the ovule in WT (h), and hug1-2 hug2-2 (i) double mutants. Asterisks indicate ovules penetrated by more than one pollen tube. Arrows indicate micropyles wherein the pollen tube enters the embryo sac. Scale bar, 100 μm.

Extended Data Fig. 3 Sperm-cell transport fails in the hug1 hug2 mutant.

(a) Successful sperm transport by the intact MGUs in WT pollen tubes that have grown out of the WT style. Insets, MGU. (b) Failed sperm transport in hug1-1 hug2-1 pollen tubes that have grown out of the WT style. Arrow, free vegetative nuclei in pollen tubes. Scale bars in A and B, 100 μm. (c, d) Failed sperm transport in hug1-2 hug2-2 pollen tubes germinated in vitro (c) and semi-in vivo (d). Scale bar, 10 μm in (c), 100 μm in (d). (e) Percentage of intact MGUs in the WT and hug1-2 hug2-2 mutant. n = 500 pollen tubes for each genotype. Data are the mean ± S.D. Two-sided Students’ t-test, ***P = 1.06 × 10−10. (f–i) Kymographs of vegetative-nucleus movement in WT (f) and hug1-1 hug2-1 (h) pollen tubes. Vertical axes represent time (every frame was equal to 5 sec, total = 30 min) and horizontal axes represent the distance along the pollen tube (total length = 229.4 μm). The short black lines on the gray background mark the trajectories of cytosolic organelles and the white line marks the trajectories of VN. In (g) and (i), three trajectories for vegetative nuclei and cytosolic organelles are marked by red lines (a distance traveled by a vegetative nucleus) and blue lines (a distance traveled by cytosolic organelles) during a given time, respectively, in the WT (g) and hug1-1 hug2-1 (i). (j, k) Speed of cytosolic organelles and vegetative nuclei marked by red or blue lines as in (g) and (i) for WT (j) and hug1-1 hug2-1 (k).

Extended Data Fig. 4 HUGs on the vegetative nucleus contribute to speed control of vegetative nuclei.

(a, b) Kymograph image showing LatB treatment disrupts MGU movement in WT pollen tubes (a) and the free vegetative nucleus in hug1-1 hug2-1 pollen tubes (b). (c, d) Kymograph image showing BDM treatment disrupts MGU movement in WT (c) and the free vegetative nucleus in hug1-1 hug2-1 (d) pollen tubes. Each image is representative of 50 assayed pollen tubes. Arrows indicate pollen-tube tips.

Source data

Extended Data Fig. 5 HUG1–GFP and HUG2–RFP fusion constructs rescue the hug1-2 hug2-2 and double mutants.

(a–c) Seed set of WT, hug double-mutant and transgenic plants expressing HUG1:HUG1–GFP, HUG2:HUG2–RFP and UBQ10:HUG2–GFP complementation cassettes in the indicated mutant backgrounds. Data are the mean ± S.D. Two-sided Students’ t-test, ***P < 0.001. The rescued lines of HUG1 also show significant different to the WT. ***P = 0.00026 (#1), ***P = 1.92 × 10−5 (#2) and ***P = 1.85 × 10−15 (#3) compared with WT. For (a), n = 466, 478, 424, 414, 426 seeds scored for genotypes from left to right. ***P = 2.01 × 10−13 (#1), ***P = 4.74 × 10−17 (#2) and ***P = 1.17 × 10−14 (#3) compared with hug1-2 hug2-2. For (b), n = 562, 492, 578, 568 and 562 seeds from left to right. ***P = 8.56 × 10−21 (#1), ***P = 2.83 × 10−19 (#2) and ***P = 3.49 × 10−21 (#3) compared with hug1-2 hug2-2. For (c), n = 3,451, 4,103, 4,098, 4,117 and 4,036 seeds scored for genotypes from left to right. Two-sided Students’ t-test, ***P = 8.19 × 10−6 (#1), ***P = 1.68 × 10−5 (#2) and ***P = 1.53 × 10−5 (#3) compared with hug1-2 hug2-2. (d) Colocalization HUG1–GFP and HUG2–RFP on the MGU in pollen co-expressing these markers. Scale bar, 5 μm. (e) HUG1–GFP fluorescence was not detectable in DAPI-stained tetrads and early microspores. Scale bar, 10 μm.

Extended Data Fig. 6 The microtubule cage is essential for HUG-cage assembly and male germ-unit organization.

(a) GFP–MAP4–MBD around the sperm nuclei in pollen tubes. Arrow, the sperm projection into the invagination of the vegetative nucleus. Scale bar, 10 μm. (c) Co-expression of GFP–MAP4–MBD and HUG2–RFP showing the microtubule cage is inside the HUG2–GFP cage. Scale bar, 5 μm. (b) Relative fluorescence intensity along the white line in (a). (c, d) Confocal imaging of GFP–MBD- and HUG1–GFP-expressing mature pollen treated with oryzalin at 25 °C and 4 °C. Scale bar, 5 μm. (e, f) Treatment of mature pollen grains with 5 μM oryzalin overnight partially disturbs HUG1–GFP localization outside the peri-germ cell membrane, causing pollen-tube branching or bulging, but does not affect the integrity of MGU. Scale bar, 10 μm. (g) Colchicine treatment of HUG1–GFP-expressing pollen. Arrow, VN-connecting filamentous structures labeled by HUG1–GFP. Scale bar, 2 μm. The mock-treated pollen is the same as that in (c, d). (h) Treatment of mature pollen grains with 1 mM colchicine disrupts HUG1–GFP localization outside the peri-germ cell membrane but does not disassemble the MGU. Asterisks indicate sperm nuclei. Scale bar, 10 μm. (i) Frequency of pollen developmental stages after supplementation to inflorescence with oryzalin for 3 d. (j) Pollen-germination rate after treatment in (i). n = 500 for each sample. Data are the mean ± S.D. Two-sided Students’ t-test, P = 0.0668 for Oryzalin-treated WT, P = 0.0585 for GFP-MAP4-MBD and P = 0.0712 for HUG1-GFP. n.s. non-significant.

Extended Data Fig. 7 HUG proteins promote assembly of microtubule cages.

(a) GFP–MAP4-MBD failed to detectably localize around the endo-plasma membrane in mature pollen from the hug1-2 hug2-2 double mutant. Asterisks in a indicate SC nuclei. Scale bar, 5 μm. (b) Expression of GFP–MBD in hug1-2 hug2-2 pollen stained with DAPI at different developmental stages. BCP, bicellular pollen. Scale bar, 5 μm.

Extended Data Fig. 8 Interaction tests between domains of HUG1.

(a) BiFC assay in Arabidopsis protoplasts showing the physical interaction between HUG1/2 and WIT1 and WIP1. YFPC, C-terminus of YFP. YFPN, N-terminus of YFP. Scale bar, 10 μm. (b) Yeast two-hybrid showing the interaction between CC1, but not between CC2_Tail.

Supplementary information

Supplementary Information (download PDF )

Supplementary Table 1. Primer list.

Reporting Summary (download PDF )

Supplementary Video 1 (download MP4 )

MGU in WT pollen tubes germinated in vitro.

Supplementary Video 2 (download AVI )

MGU in hug1-1 hug2-1 pollen tubes germinated in vitro.

Source data

Source Data Fig. 4 (download PDF )

Unprocessed western blots and/or gels.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chang, S., Ali, I., Zhou, PM. et al. Kinesins control male germ unit assembly for sperm delivery in Arabidopsis. Nat. Plants 11, 1798–1809 (2025). https://doi.org/10.1038/s41477-025-02084-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41477-025-02084-9

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing